With the advent of clinical organ transplantation as an accepted method of treatment for various diseases, the need for preservation of cadaver organs became established. The requirements of a media that will support organ perfusion were partially accomplished by the use of cryoprecipitated plasma (CPP). Thus, the problems associated with blockage of capillaries by lipoproteins, fibrin aggregates, and the like, were to a certain degree prevented. There are, however, several disadvantages related to the use of CPP: (a) it is difficult to standardize and store; (b) it must be thawed and filtered immediately before use; and (c) there is a risk of transmission of hepatitis in the use of unstored plasma. Most important, it is not reliable for long-term perfusion since renal function does not reliably and immediately reappear upon transplantation of kidneys preserved for more than 48 hours.
A number of efforts have been made to eliminate some of these disadvantages. In particular, the use of synthetic plasma derivatives and albumin solutions have been tried. Such solutions theoretically should contain little aggregated materials, i.e., lipoproteins and partially polymerized fibrinogen and since they can be stored there is little risk of hepatitis. However, for unknown reasons these materials are not as satisfactory as plasma. In applicants' laboratory at the University of Minnesota Medical School, canine kidneys perfused with albumin demonstrated only 50 percent survival after 48 hours preservation. Furthermore, only 25 percent of the kidneys perfused with plasmanate survived after the same preservation period.
One possible explanation why CPP is superior to the other plasma derivatives for organ perfusion is the trace materials within plasma which are necessary for metabolic maintenance of the hypothermic kidney. Such materials may be removed by the processing of albumin or plasmanate. At the same time it may be possible that the problem with CPP is related to the failure to remove by freezing all of the precipitable and denaturable elements within the plasma.
The picture for shock is even less clear. Upon storage, plasma and blood lose their effectivenss as plasma expanders in hypovolemic and endotoxic shock. Serum, the protein fluid remaining after the polymerization of fibrinogen, is of little value in organ perfusion and may be even toxic, whereas, in shock, even saline is more effective than serum, suggesting that in the clotting of blood harmful pharmacological active factors may be generated.
The deficiencies in the above mentioned preparations led to the investigation and development of the method of the present invention for (1) removing fibrinogen from plasma without its polymerization to fibrin and leaving prothrombin at pretreatment levels, (2) removing the plasminogen-plasmin proteolytic enzyme system, (3) without generation of toxic factors nor removal of biologically essential support factors, and (4) allowing for long-term storage of plasma without loss of its biological support properties.
A series of investigations with various natural and synthetic poly-silica compounds, resulted in the discovery that a synthetic fumed colloidal silicon dioxide could be used to treat plasma removing fibrinogen without polymerization, and preventing the accumulation of pharmacologically active toxic split products. Subsequent research led to the discovery that in addition the proteolytic enzyme system plasminogen-plasmin is also removed by fumed silica treatment. Plasma treated and processed in this manner has been extensively tested in both animal and human models and evaluated for efficacy for organ preservation, shock, and for retention of its biological support properties following long-term storage (1 year).
Blood contains plasminogen, the inactive precursor of the potent proteolytic enzyme plasmin. Under ordinary conditions such as those involved in the preparation of plasma from citrated whole blood, plasminogen is not activated in any detectable quantities since there are inhibitors in plasma which can act to block activation by kinases as well as inhibitors that can block the action of the enzyme plasmin. However, on long-term storage of plasma there apparently is some activation of plasminogen to plasmin (up to 5 percent) and the subsequent proteolysis of fibrinogen, immunoglobulins, and other proteins. With plasma develops visible aggregates which are presumably products of partially degraded fibrinogen and immunoglobulins. This problem is largely circumvented by the outdating of plasma, resulting in discarding and loss of a valuable resource.
In the classic Cohn alcohol fractionation of human plasma, the plasminogen is concentrated and freed of its inhibitors in the fraction process. The result is that fractions I, II, III of the Cohn procedure contain much greater quantities of plasminogen than are present initially in plasma. The presence of high concentrations of plasminogen, if activated, leads directly to the degradation of fibrinogen to form the toxic so-called split products. In addition, it has been demonstrated that the plasmin system can partially degrade the immunoglobulins, a process which leads to formation of molecular aggregates.
Immunoglobulins acted upon by plasmin when injected into the circulation are eliminated very rapidly when compared to normal, unaltered immunoglobulins. The net effect is to prevent the attainment of high blood levels that are necessary in treating bacterial, toxic states and viral diseases. In particular, the plasmin altered immunoglobulin when administered to immune deficiency patients intravenously produce anaphylactoid-like reactions, thereby eliminating a potentially effective method of treating such patients.
A major obstacle to the preparation of potent solutions of purified immunoglobulins that can be safely administered intravenously to patients to achieve high blood levels in treating immune defects and life threatening infections has been the failure to either eliminate or prevent the aggregation of immunoglobulins during purification. Aside from the fact that the universally employed Cohn alcohol method of plasma protein fractionation may irreversibly denature some plasma proteins, the presence of the plasma proteolytic enzyme plasminogen has been demonstrated to attack, and particularly degrade by its proteolytic activity, immunoglobulins, particularly the IgG class. The IgG immunoglobulins that have been attacked by plasmin form molecular aggregates which have been implicated in the activation of the kinen and complement systems and further, when their aggregated solutions are administered intravenously to patients with immune deficiency, precipitate anaphylactoid systemic reactions. In addition, these partially degraded aggregated IgG preparations are rapidly eliminated from the circulation thereby significantly reducing the effectiveness of specific antibody in conferring protection to toxic states resulting, for example, form diphtheria toxin, and protection against infection. Thus, the hoped for goal of achieving high effective blood levels of a biologically active antibody to toxins, viral or bacterial organisms have not been attained to date.